Sex hormones differently regulate lipid metabolism genes in primary human hepatocytes

Background Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is higher in men than in women. Hormonal and genetic causes may account for the sex differences in MASLD. Current human in vitro liver models do not sufficiently take the influence of biological sex and sex hormones into consideration. Methods Primary human hepatocytes (PHHs) were isolated from liver specimen of female and male donors and cultured with sex hormones (17β-estradiol, testosterone and progesterone) for up to 72 h. mRNA expression levels of 8 hepatic lipid metabolism genes were analyzed by RT-qPCR. Sex hormones and their metabolites were determined in cell culture supernatants by LC-MS analyses. Results A sex-specific expression was observed for LDLR (low density lipoprotein receptor) with higher mRNA levels in male than female PHHs. All three sex hormones were metabolized by PHHs and the effects of hormones on gene expression levels varied depending on hepatocyte sex. Only in female PHHs, 17β-estradiol treatment affected expression levels of PPARA (peroxisome proliferator-activated receptor alpha), LIPC (hepatic lipase) and APOL2 (apolipoprotein L2). Further changes in mRNA levels of female PHHs were observed for ABCA1 (ATP-binding cassette, sub-family A, member 1) after testosterone and for ABCA1, APOA5 (apolipoprotein A-V) and PPARA after progesterone treatment. Only the male PHHs showed changing mRNA levels for LDLR after 17β-estradiol and for APOA5 after testosterone treatment. Conclusions Male and female PHHs showed differences in their expression levels of hepatic lipid metabolism genes and their responsiveness towards sex hormones. Thus, cellular sex should be considered, especially when investigating the pathophysiological mechanisms of MASLD. Supplementary Information The online version contains supplementary material available at 10.1186/s12902-024-01663-9.


Fig. S3
Fig. S3 Chromatograms and mass spectra of sex hormone metabolites.Primary human hepatocytes (PHHs) from female and male donors were cultured with 50 µM 17β-estradiol, testosterone or progesterone for 5 h.Cell culture media were collected and analyzed by LC-MS after cleavage of phase II metabolites.The following sex hormone metabolites were identified: A estrone, B 2-methoxyestrone, C 2hydroxyestradiol, D hydroxytestosterone, E 11-ketotestosterone, F dihydrotestosterone, G hydroxyprogesterone.

Fig. S5
Fig. S5 Influence of 17β-estradiol, testosterone and progesterone on UGT2B15 mRNA expression levels of primary human hepatocytes (PHHs) of different sex.PHHs were isolated from liver tissues of female (A, C, E) and male (B, D, F) donors, cultured with PHH starving medium supplemented with 10 nM 17β-estradiol (A, B), 40 nM testosterone (C, D) or 70 nM progesterone (E, F) for up to 72 h and mRNA expression levels of UGT2B15 (UDP-glucuronosyltransferase 2B15) were analyzed by RT-qPCR.RPL13A (ribosomal protein L13a), EEF2 (eukaryotic elongation factor 2) and RPS18 (ribosomal protein S18) served as reference genes.Individual fold change values are displayed as dots and cubes, bar graphs display means ± SEM, n = 5 per sex, paired t test, p < 0.05 (*).

Table S1
Donor data for PHH suspension cultures

Table S2 Primer specifications Gene Assay ID 1 or primer sequence 2 (fwd / rev)
1 Primers purchased from Qiagen 2 Primers purchased from Biomers

Table S3
UP-LC gradient program

Table S5
Sex-specific mRNA expression levels of PHHs immediately post-

Table S6
Effects of 17β-estradiol on cultured primary human hepatocytes (PHHs) of different sex

Relative mRNA expression in female PHHs Relative mRNA expression in male PHHs
Relative mRNA expression values are displayed as geometric means of individual fold change values (SEM).

Table S7
Effects of testosterone on cultured primary human hepatocytes (PHHs) of different sex

Table S8
Effects of progesterone on cultured primary human hepatocytes (PHHs) of different sex Relative mRNA expression values are displayed as geometric means of individual fold change values (SEM).